REGULATION AND ROLE OF IL-7 PRODUCTION IN HIV-1 INFECTION

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From the Department of Microbiology, Tumor and Cell Biology Karolinska Institutet, Stockholm, Sweden

REGULATION AND ROLE OF IL-7 PRODUCTION IN HIV-1 INFECTION

Pham Hong Thang

Stockholm 2011

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All previously published papers were reproduced with permission from the publisher.

Published by Karolinska Institutet. Printed by Universitetsservice US-AB

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In memory of my father

to my mother and my parents in law

and to Mai, Dinh Duy and Duc Anh

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ABSTRACT

The concentration of interleukin-7 (IL-7) in human serum is elevated in various clinical conditions associated with lymphopenia, including HIV-1 infection. IL-7 is an essential factor for T cell differentiation and survival, and it was suggested that high serum IL-7 concentration may represent a homeostatic response to T cell depletion, which may promote T cell regeneration.

In order to increase our understanding on the regulation of IL-7 production, we investigated specimens from HIV-1 infected patients during chronic infection and in long term non-progressors (LTNPs). Serum IL-7 levels correlated with T-cell depletion in HIV-1 infected individuals. In some patients, we observed that serum IL-7 decreased upon progression to AIDS, suggesting a role for IL-7 in T-cell maintenance in sporadic cases. Interestingly, IL-7 levels were significantly lower in stable LTNPs than in patients who lost the LTNP status in a 3-year follow-up (P<0.001), indicating that serum IL-7 concentration might be a valuable marker for maintenance of the LTNP status.

The number of CD8+CD28- T cells increases significantly during aging and during HIV-1 infection.

These cells have a reduced expression of the IL-7 receptor alpha (IL-7RD), as compared to CD8+CD28+ T cells. As CD8+CD28- T cells have been associated with dendritic and T cell suppression, we analyzed whether an increase in CD8+CD28- T cell numbers during HIV-1 infection could lead to impaired T cell responses. Peripheral blood CD8+CD28- T cells of both HIV-infected and non-infected individuals promoted dendritic cell activation. The CD8+CD28- T cell accumulation during HIV-1 infection may thus contribute to inflammatory reactions and immune activation.

Stromal cells and intestinal epithelial cells are known to produce IL-7. The mechanisms and cellular factors regulating IL-7 production are still unclear. We assessed whether IL-1E and IFN-J, cytokines produced during inflammatory conditions, may impact on IL-7 production. We used human intestinal epithelial cells (DLD-1 cell line) and bone marrow stromal cells (HS27 cell line) to evaluate IL-7 production at the mRNA and protein levels. To assess whether treatment of HS27 cells with IL-1E and/or IFN-J leads to changes in the gene expression of cytokines, Toll-like receptors (TLRs) and chemokines, we analysed gene expression profiles using the whole-genome microarray Human Gene 1.0 ST. We found that IFN-J enhanced the expression of IL-7 protein and mRNA (P<0.001) in both cell lines. IL-1E treatment led to a significant down-regulation (P<0.001) of IL-7 mRNA expression in both cell lines. The gene profiles revealed dramatic changes in expression of cytokines and their receptors, of IFN regulatory factors (IRF-1 and 2) and of important chemo-attractants for T cells. The microarray results were verified by additional methods. Our results were discussed in the setting of inflammation and T-cell survival in the gut compartment during HIV-1 infection where stromal and epithelial cells may produce factors that contribute to impaired IL-7 homeostasis and homing of T cells.

It was previously reported that IL-7 might stimulate T cell activation and CD95 mediated T cell apoptosis. HIV-1 infection leads to B cell abnormalities including increased apoptosis via the CD95 death receptor pathway and loss of memory B cells. Here we present a novel mechanism that can lead to increased B cell apoptosis in the presence of high IL-7 concentration. T cells cultured with IL-7 induced high CD95 expression on resting B cells together with an increased sensitivity to CD95 mediated apoptosis. As the mediator molecule responsible for B cell priming to CD95 mediated apoptosis we identified the cytokine IFN-J that T cells secreted in response to IL-7. In the serum of HIV-1 infected individuals IL-7 and IFN-ȖOHYHOVZHUHLQFRUUHODWLRQDQGWKHOHYHORIERWK

cytokines correlated with CD95 expression on circulating B lymphocytes in non-viremic individuals. These results indicate a potential link between IL-7 and the increased B cell apoptosis observed in HIV-1 infected individuals.

In conclusion the results presented in this PhD thesis highlight mechanisms of regulation of IL-7 production dependent on the number of circulating T cells and on the exposure of IL-7 producing cells to high levels of inflammatory cytokines. We also present data on the role of IL-7 in regulating CD95 expression and CD95 mediated apoptosis on B cells through IFN-J produced by T cells; the impact of this finding on the outcome of IL-7 therapy during HIV-1 infection will be verified by ongoing clinical studies.

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LIST OF PUBLICATIONS AND MANUSCRIPT

I. Fluur C, Rethi B, Thang PH, Vivar N, Mowafi F, Lopalco L, Foppa CU, Karlsson A, Tambussi G, Chiodi F. Relationship between serum IL-7 concentrations and lymphopenia upon different levels of HIV immune control. AIDS. 2007 May 11;21(8):1048-50.

II. Vivar N, Thang PH, Atlas A, Chiodi F, Rethi B. Potential role of CD8+CD28- T lymphocytes in immune activation during HIV-1 infection.

AIDS. 2008 May 31;22(9):1083-6

III. Thang PH, Ruffin N, Brodin D, Rethi B, Cam PD, Hien NT, Lopalco L, Vivar N, Chiodi F. The role of IL-1beta in reduced IL-7 production by stromal and epithelial cells: a model for impaired T-cell numbers in the gut during HIV-1 infection. J Intern Med. 2010 Aug;268(2):181-93.

IV. Sammicheli S, Thang PH*, Dang VPL*, Ruffin R, Pensieroso P, Vivar N, Hejdeman B, Chiodi F and Rethi B. IL-7 promotes CD95-induced apoptosis in B cells via the IFN-Ȗ67$7SDWKZD\. (Submitted)

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LIST OF ABBREVIATIONS

AIDS Acquired immune deficiency syndrome

APC Antigen presenting cell

ART Antiretroviral therapy

Bcl-2 B-cell lymphoma 2

BM Bone marrow

CCR C-C chemokine receptor

CFSE Carboxyfluorescein succinimidyl ester CD Cluster of differentiation

cDNA Complementary DNA

CTL Cytotoxic T cell CXCL12 Chemokine C-X-C motif ligand 12

CXCR C-X-C chemokine receptor

DC Dendritic cell

DLD-1 Human intestinal epithelial cell line DLD-1

DNA Deoxyribonucleic acid

ELISA Enzyme-linked immunosorbent assay FRC Fibroblast reticular cells

GALT Gut-associated lymphoid tissue

GI Gastrointestinal

GM-CSF Granulocyte-macrophage colony-stimulating factor HAART Highly active antiretroviral therapy

HIV Human immunodeficiency virus

HS27 Human bone marrow stromal cell line HS27

HTLV Human T lymphotropic virus

ICAM-1 Inter-cellular adhesion molecule 1

IFN Interferon

IL Interleukin

IL-7R IL-7 receptor

IRF IFN regulatory factor

JAK Janus kinase

LPS Lipopolysaccharides

LTNP Long-term non-progressor

mRNA Messenger RNA

NK Natural killer cell

PBMC Peripheral blood mononuclear cell

PCR Polymerase chain reaction

PCs Plasma cells

RNA Ribonucleic acid

SIV Simian immunodeficiency virus

STAT Signal transducer and activator of transcription factor T reg T regulatory cells

TLR Toll-like receptor

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1 INTRODUCTION

1.1 HIV AND THE PATHOGENESIS OF HIV INFECTION

1.1.1 Human immunodeficiency virus - HIV

In 1981, a new disease syndrome manifested in some previously healthy homosexual men in the United States characterized by unusual infections and cancers such as Pneumocystis carinii pneumonia and Kaposi’s sarcoma. A marked reduction in cluster of differentiation (CD)4+ T cells was the common immunological dysfunction which characterized this group of patients. This new syndrome was named acquired immune deficiency syndrome (AIDS) (1). Two years later, the causative agent of AIDS was identified first by a group of scientists at the Pasteur Institut in Paris (2) and then by groups in the United States (3, 4). The causative agent of this syndrome was a retrovirus initially named human T lymphotropic virus type III (HTLV-III) until the new name human immunodeficiency virus (HIV) was agreed on by a panel of experts in the field.

HIV is a member of the Lentivirus sub-family in the Retroviridae family. The family is named Retrovirus since the viral ribonucleic acid (RNA) genome is transcribed into deoxyribonucleic acid (DNA) within the host cell using the viral enzyme reverse transcriptase (RT). This viral DNA is then transported to the nucleus and integrates into the cellular chromosome. The virus genome can be silent in the host cell until it is transcribed and the viral replication initiated.

HIV quickly spreads around the world. According to a report published in 2010, Joint United Nations Programme on HIV/AIDS (UNAIDS) estimated that there were 33.3 million adults and children leaving with HIV/AIDS world-wide, 2.6 million people newly infected with HIV in 2009 and 2.1 million AIDS-related deaths among adults and children in 2009 (5).

There are two types of HIV named HIV-1 and HIV-2 and the distinction between the two types is based on virus genetic differences. HIV-2 was identified two years after the discovery of HIV-1 (6). The genetic difference between HIV-2 and HIV-1 is about 40%. HIV-1 can also be divided into different groups M (main), O (outlier), and N (new or non M or O) and the genetic difference of virus groups is at least 30% from one to another, with variations mainly in the envelope genes (7). Recently, a new HIV-1

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group was identified, named P, that is closely related to gorilla simian immunodeficiency virus (SIVgor) (8). The HIV-1 M group is further divided into subtypes (or clades) A-D, F-H, J, K and the subtypes differ by 15-20% in the genes (7).

The genomic recombination of different HIV-1 subtypes created different HIV-1 recombinant viruses called circulating recombinant forms (CRFs) (9). Up to date more than 40 CRFs have been identified (www.hiv.lanl.gov). Of note, the original subtype E and I have been identified as recombinant subtypes. Virus recombination can occur after co-infection or super-infection of the same cell by two or more different virus subtypes (10).

HIV-1 caused the majority of HIV infection and distribution world-wide. By contrast, HIV-2 was primarily isolated from patients in West Africa although this virus type was also detected in individuals in other parts of the world including Europe, the United States, South America, and India. However, most of the HIV-2 cases detected in Europe and in the United States were from African migrations (11).

HIV-2 infection has a less pathogenic nature as compared to HIV-1 infection. There are several features that could possibly explain for the HIV-2 properties. During HIV-2 infection, lower viral load is generally detected in blood and genital fluids than the corresponding values found in HIV-1 infected subjects (12-14); the envelope glycoprotein (gp) 105 has a reduced affinity for the cell surface receptors, as compared to HIV-1 gp120 (15); patients infected with HIV-2 display a reduced level of immune activation (16, 17), low level of T cell apoptosis (16, 18) and lower CD8+ T cell cytotoxic immune response as compared to patients infected with HIV-1 (19).

1.1.2 HIV transmission

There are three main routes of HIV transmission, sexual contact, blood and blood products, and mother-to-child transmission. The transmission through the blood route has been proven to be most effective (20-24). However, HIV transmission through unprotected sexual intercourse is widely propagated. HIV transmission from mother-to- child can take place in utero, at delivery and during breast feeding. Without antiretroviral treatment (ART) the transmission from mother to child is believed to occur in 25-40% of pregnancy and delivery cases. The rate of transmission can be significantly reduced to < 10% or even to <2% with ART treatment of the mothers and

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During primary HIV-1 infection, the virus infects a large number of CD4+ T cells. The HIV-1 virions replicate and spread very efficiently and the CD4+ T cells decrease sharply. During the acute phase of the HIV-1 infection, patients can experience influenza-like symptoms including fever, myalgia and skin rash. However, in most of cases those symptoms are minor and can be confused with other diseases or not at all recognized by the patient (25-28).

HIV-1 infects the host cell through the binding of the viral envelope gp120 to a high affinity receptor present at the cell membrane, the CD4 receptor. The CD4 receptor is expressed on T cells. macrophages, monocytes and dendritic cells (DCs) (29, 30). The HIV-1 gp120 molecules interacts with the CD4 receptors on the target cell and mediates the virus entry by fusing together the viral and the cell membranes; the C-C chemokine receptor (CCR) CCR5 or C-X-C chemokine receptor (CXCR) CXCR4 molecules act as co-receptors during this process. The interaction of the complex composed of CD4 and gp120 with a specific co-receptor triggers further conformational changes in the envelope glycoprotein complex. That process leads to the exposure and insertion of the hydrophobic gp41, the fusion peptide of the virus, into the membrane of the target cell.

HIV-1 isolates can preferentially infect either macrophages or T cells and accordingly they are named as macrophage tropic (M-tropic) or T cell tropic (T-tropic) isolates. A macrophage-tropic HIV-1 virus uses CCR5 receptor as co-receptor to attach and infect the cell and is accordingly classified as a R5 virus. HIV-1 viruses with the property of infecting T cell lines are called X4 viruses and infect T cells using CXCR4 as co- receptor. The X4 viruses are more cytopathic than the R5 viruses (31). Individuals who lack CCR5 expression on immune cells due to PXWDWLRQLQWKH&&5ǻ gene are resistant to R5 virus infection but are still susceptible to infection with X4 viruses.

Some other chemokine coreceptors (e.g., CCR3, CCR2b) are known to act as primary or secondary attachment sites for both HIV-1 and HIV-2 isolates, but are not commonly involved in infection (31).

Some studies have shown that R5 viruses are generally observed in the blood during acute or early infection. However, during advanced disease progression and AIDS, approximately 50% of patients carry cytopathic X4 viruses in blood (32, 33). R5 viruses infect preferentially CCR5+CD4+ T cells in the gastrointestinal (GI) tract and R5 viruses can easily infect macrophages and DCs (7).

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1.1.3 Immune response in HIV-1 infection

HIV-1 infection targets cells of the immunological system and renders them dysfunctional. The immunopathological manifestations of HIV-1 infection are generally a gradual reduction of CD4+ T cells in the peripheral blood as well as in the lymphoid tissues and gut-associated lymphoid tissue (GALT); enhanced B cells proliferation and hypergammablobulinemia – features which may reflect immune activation occurring during chronic infection. An overt stage of immune deficiency is often accompanied by opportunistic infections and malignancies like Kaposi’s sarcoma.

The mechanisms through which HIV-1 induced immune activation is established over time leading to destruction of the immune system are depicted in Fig. 1.

1.1.3.1 T cells

CD4+ T cells loss is the main pathogenic feature of HIV-1 infection. At the acute phase of HIV-1 infection, there is a dramatic depletion of CD4+ T cells which can be detected in peripheral blood; thereafter the number of CD4+ T cells rebounds to certain levels after the initial HIV-1 burst decreases to a set point in the host body. Without any treatment, the CD4+ T cells are gradually lost during the following years of HIV-1 infection.

In SIV infection in macaques within a few days from infection, the virus rapidly migrates to the GALT where it induces a massive depletion of memory CD4+ T cells in the intestinal lamina propria (34, 35). The same picture of CD4+ T cells depletion in the GALT as the one occurring in SIV infected macaques was observed in humans at the early stage of HIV-1 infection (36-38).

Depletion of CD4+ T cells happens not only in peripheral blood and GALT, but also at other mucosal sites and in the lymph nodes. Studies in SIV and HIV-1 infection also demonstrated that the CD4+ T cells depletion is less pronounced in peripheral blood and lymph nodes as compare to GALT (37, 39).

There are some potential factors involved in the loss of CD4+ T cells in HIV-1 infection. The direct cytopathic effects of HIV-1 on CD4+ T cells and progenitor cells lead to cell death (40). HIV-1 induced apoptosis of the target cells and it was shown that HIV-1 encoded proteins induced apoptosis of infected and uninfected cells (41-43).

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tissue reduces the production of new cells (45, 46). CD4+ T cells loss is the primary reason for occurrence of the opportunistic infections and cancers associated with HIV-1 infection.

CD8+ T cells so called cytotoxic T cells (CTLs) have been found to be increased in percentage as well as in absolute number in HIV-1 infection (47) and CD8+ specific T cells are distributed in different anatomical compartments in both humans and macaques. CD8+ specific T cells appear to be involved in the control of HIV-1 replication (48) and in vitro CD8+ T cells can control HIV-1 infection by inhibiting HIV-1 replication (49). Recent evidence also showed that early HIV-1 specific CD8+ T cell responses contribute to reduce HIV-1 viremia in plasma during the acute phase of HIV-1 infection (50). CD8+ T cells are also involved in adaptive immune function by killing the virus infected cells. CD8+ T cells produce some soluble factors such as RANTES (Regulated upon Activation, Normal T-cell Expressed, and Secreted), macrophage inflammatory protein (MIP)-1D and MIP-1E (51), and additional important cytokines like interferon gamma (IFN)-J and tumor necrosis factor alpha (TNF)- DIt is important to underline that CD8+ T cells activities can be detrimental if they lead to lysis of autologous uninfected CD4+ T cell and antigen presenting cells (APCs). It was shown that the expression of the programmed death 1 protein (PD-1) on HIV-1 specific CD8+ T cells can reduce their cytotoxic function (53).

1.1.3.2 B cells

Another important component of HIV-1 pathogenesis is the damage occurring to B lymphocytes, which can be measured by altered phenotype and composition of different B cells populations, increased apoptosis of these cells and an abnormal pattern of activation (54). Phenotypic and functional alterations on B lymphocytes are often observed in chronically infected patients, but alterations of B cells are already detected during the acute phase of HIV-1 infection (55). A decline of memory B cells has previously been reported to occur in both children and adults infected with HIV-1 (56, 57); these cells are responsible for mounting and maintaining an adequate serological response to antigens previously encountered in life through natural infection or vaccination. The decline in B cells carrying immunological memory correlated to loss of antibody titers to measles, tetanus, and pneumococcal antigens, a process which already began during primary HIV-1 infection. The consequences of loss of memory B

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Figure 1. HIV-1 infection induces immune activation and destruction of the immune system. Due to HIV-1 infection, innate and adaptive immune cells become activated by different factors like viral proteins, microbial components translocated from the GALT and host immune responses. Activated cells release proinflammatory cytokines (IFN, TNF, IL-1, IL-6), which in turn, together with HIV-1 replication and host immune responses, contribute to chronic immune activation. Chronic immune activation increases HIV-1 replication and leads to immune cell exhaustion, finally causing damage to the immune system.

cells are not fully understood; the loss of memory B cells during HIV-1 infection may be mediated by up-regulation of CD95 (Fas) receptor and increased susceptibility to CD95 mediated apoptosis (55).

1.2 APOPTOSIS

Apoptosis, or programmed cell-death, provides a mechanism for removal of senescent cells without evoking inflammatory responses. Two main pathways have been identified for execution of apoptosis: 1) the intrinsic apoptotic pathway, also defined as the mitochondrial pathway is receptor-independent, and requires mitochondrial

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pro- and anti-apoptotic proteins. Caspase activation and cleavage of specific cellular substrates occur as result of both the intrinsic and extrinsic pathways, ultimately leading to chromatin condensation and DNA fragmentation. The process of apoptosis is completed when the apoptotic cells are removed by phagocytosis. Due to efficient phagocytosis, few apoptotic cells are found in healthy tissues despite a constant homeostatic turnover of senescent cells.

Different apoptotic pathways have been described to date, many of which overlap each other at the molecular level. The permeabilization of the mitochondrial outer membrane (MOMP) is a pivotal point of mitochondrial apoptosis pathway which triggers caspase activation resulting in irreversible events of cell apoptosis. This intrinsic apoptotic pathway involves the regulation of different important proteins of the B-cell lymphoma (Bcl)-2 family, which comprises, among others, the anti-apoptotic proteins Bcl-2, Bcl- xL and Bim and the pro-apoptotic proteins Bax and Bak (58). Extrinsic apoptosis can be initiated through activation of a number of membrane bound receptors; among them the CD95 protein which is expressed as a transmembrane receptor (59) and is ubiquitously expressed in the majority of cell types. The natural ligand for CD95 is CD95 ligand (CD95L) mainly expressed by activated T-cells, NK cells and macrophages. An important molecular component in the structure of death receptors is a cytoplasmic domain of approximately 80 residues named death domain which plays an important role for recruitment of molecules which initiate intracellular signalling leading to apoptosis.

CD95/CD95L interactions have been proposed to be necessary to down-regulate the number of reactive cells during contracting phase of immune responses (60). Aberrant apoptosis of T cells during HIV-1 infection leads to immunosupression and susceptibility to opportunistic infections (61). Studies on the impact of viral infection on the GALT have demonstrated that apoptosis is a pivotal mechanism for HIV-1 driven destruction of mucosal CD4+ T cells. In addition, high levels of CD95/CD95L expression and CD95-mediated apoptosis were detected in lamina propria T cells (35).

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1.3 INTERLEUKIN-7 AND IL-7 RECEPTOR

1.3.1 Interleukin-7

The Interleukin (IL)-7 belongs to the type 1 cytokines of the hematopoietic family. IL-7 is a non-redundant cytokine with important roles in lymphocytes development and survival and maintenance of peripheral lymphocytes homeostasis. IL-7 was first discovered in 1988 as a novel growth factor for precursor murine B cells produced in vitro from bone marrow derived stromal cells (62). Additional studies conducted later on showed that IL-7 has an effect on growth and differentiation of immature and mature T cells and fetal thymocyte clones (63, 64). IL-7 was demonstrated to be required for the survival and proliferation of mature and naive peripheral T cells (65).

Recently, an in vitro study was conducted by culturing peripheral blood mononuclear cell (PBMCs) in presence or absence of IL-7, followed by infection with an HIV-1 R5 strain (66). Interestingly, IL-7 increased the density of CXCR4 receptor at the CD4+ T cell surface and induced the switch of HIV-1 R5 virus to X4 virus.

Human IL-7 gene spans 6 exons located on the chromosome 8q12-13 encoding for a protein of 177 amino acids (aa) with molecular weight of 25 kilo Dalton (kD) in active form (67). Different IL-7 isoforms, defined as alternative splice variants, have been described; the different isoforms have been mapped in different human tissues suggesting an important role for these different isoforms in different disease conditions (68).

IL-7 is produced by non-lymphoid cells, and IL-7 messenger RNA (mRNA) has not been detected in lymphocytes during physiological conditions. In humans, different cell types are known to produce IL-7 and among them the IL-7 main source are stromal cells in the bone marrow (69, 70), intestinal epithelial cells (71, 72) and fibroblastic reticular cells (73). In addition, keratinocytes (74), peripheral blood DCs (75), follicular DCs, smooth muscle cells and endothelial cells (76) were also found to produce IL-7. Recently, a study showed that stimulation through Toll-like receptors (TLRs) of liver hepatocytes can induce production of IL-7 from hepatocytes (77). This mechanism of regulation via TLRs triggering appears to be unique for hepatocytes and the production of IL-7 by hepatocytes stimulated with lipopolysaccharides (LPS) appears to be transient. A study conducted ex- vivo has shown that IL-7 production by bone marrow stromal cells derived from HIV-1

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before ART (78). IL-7 was also shown to be produced by a small population of mice DCs regulating the niche size of CD4+ T cells in vivo (79).

How IL-7 is constitutively produced, and the mechanism for regulation of IL-7 production is yet not understood in detail. IFN-J has been shown to up-regulate the IL-7 production in human intestinal epithelial cells through IFN regulatory factors 1 and 2 (IRF-1, 2) via an IFN regulatory factor element (IRF-E) acting on the 5’ flanking region of the human IL-7 gene (80). Other factors like transforming grow factor-E (TGF-E (81) and flagellin (82) have been reported to negatively modulate the IL-7 production. On the other hand, IL-1 and TNF-D have been shown to up-regulate IL-7 production from stromal cells (83). Recently, a study showed that in vivo, commensal microflora drives IFN-J production by lymphocytes, which in turn promotes IL-7 production from intestinal epithelial cells and the survival of IL-7-dependent lymphocytes. Interestingly, the combination of IFN-J with the commensal microflora promotes a steady-state IL-7 production in the intestine (84).

IL-7 is considered as an obligate survival factor for several subsets of progenitor and mature lymphoid cells. Mutations in the IL-7 gene or its receptor complex in mice resulted in impaired IL-7 production and IL-7 mediated signaling; upon these conditions the mice became severely lymphopenic, with T cell depletion at both primary and peripheral lymphoid sites and with T cell numbers decreased 10–20-folds (85). The absence of IL-7 triggered apoptotic processes in IL-7 dependent cells, as shown by increased annexin V binding to the cell membrane and DNA fragmentation (86). More recently it has been shown that IL-7 is required for homeostatic survival of peripheral T lymphocytes. Under T cell deficient conditions, naïve T cells were able to expand in mice lacking other cytokines, such as IL-4 or IL-15, but not in mice lacking IL-7 , and the same was shown for memory CD+8 T cells (87).

1.3.2 IL-7 receptor

The IL-7 receptor (IL-7R) belongs to the cytokine receptor family. The IL-7R consists of two components: the IL-7 receptor alpha chain (D) also named CD127 and a common gamma chain (Jc) (CD132) which is shared by the receptors for IL-2, IL-4, IL-7, IL9, IL-15 and IL-21. The cytoplasmic domains of the IL-5Į DQGȖFare required for STAT5A/B activation which is followed by signal transduction. Studies conducted in mice have shown that IL-5Į DQGȖF-deficient mice presented with similar features

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characterized by diminished T cell numbers and impaired lymphocyte development (88). These data indicate that both IL-5Į DQGȖFDUHHVVHQWLDOto mediate the biological effects of IL-7 on the target cells.

The IL-7RD chain is expressed on different cell types. In human hematopoietic cells, IL-7RD is expressed on developing T and B cells, mature T cells (both naïve and memory T cells), natural killer cell (NK) and DCs. Other human cell types such as intestinal epithelial cells, endothelial cells, bone marrow stromal cells, cancer cells of colorectal, lung, and breast origin also express the IL-7RD.

IL-7RD gene is located in human chromosome 5p13, which contains 8 exons; the IL- 7RD gene encodes the IL-7RD proteins which is comprised of 439 aa and has a molecular weight of 49.5kD. The IL-7RD is a glycoprotein trans-membrane receptor including a single 25 aa transmembrane domain and a cytoplasmic tail of 195 aa.

IL-7RD expression on cell surface is regulated by different factors. It has been shown that the IL-7RD expression is increased by glucocorticoids in T cells and cell lines (89), by TNF in mice T cells, by IFN-D and IFN-E in mice and human cell lines (90). The expression of IL-7RD in T cells is suppressed by different inhibitors. The expression of the IL-7RD is suppressed by its ligand IL-7 (91) and also by some of the cytokines belonging to the common Jc receptors IL-2, IL-4, IL- 15 and other pro-inflammatory and anti-inflammatory cytokines including IL-6 (91, 92). The T cell receptor (TCR) when activated also inhibits the IL-7RD expression (89, 93). The expression of IL-7RD on T cells is down-regulated during HIV-1 infection (94, 95). It has been shown that soluble HIV-1 Tat protein removes the IL-7RD from the surface of resting CD8+ T cells. HIV-1 Tat protein targets IL-7RD for degradation via the proteasome leading to reduced IL-7 signaling and impaired CD8+ T cell proliferation and function (95, 96).

When IL-7RD is expressed in cells it binds to its specific ligand IL-7 and will induce cell survival and proliferation. However, over expression of IL-7RD is also associated with some negative effects such as induction of inflammatory bowel disease (97) and induction of lymphoma in transgenic mice (98). On the other hand, studies conducted in gld mice that lacked the IL-7RD expression, or where the IL-7RD expression was blocked, showed a pattern of inhibition of T cell proliferation and survival (99).

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1.3.3 IL-7/IL-7R signaling pathways

When IL-7 binds to its specific receptor, different signaling pathways linked to the IL-7 receptor are activated in order to obtain different biological effects in the target cells (Fig. 2).

1.3.3.1 JAK-STAT pathways

- JAK3: Janus kinase (JAK) 3 is a protein belonging to the family of intracellular tyrosine kinases. JAK3, which is recognized as the first step in the signal transduction cascade from the IL-7 receptor, is constitutively associated with the carboxy-terminal region of the ȖF FKDLQ receptor. In humans, mutations in the JAK3 gene result in a disease similar to the XSCID (X-linked Severe Combined Immune Deficiency) caused E\ PXWDWLRQ LQ ȖF FKDLQ (100). The main function of the JAK3 signaling pathway is to protect the cell from apoptotic death.

- JAK1: JAK1 is associated with the IL-5Į chain and phosphorylated following IL-7 binding to the IL-7RD. The protein tyrosine kinase Pyk2, which is related to the focal adhesion kinase, was shown to be associated with JAK1 and to play a role in survival of a thymocyte cell line (101). JAK1 activity is required for the IL-7 mediated inhibition of TGF-ȕ production and signaling by fibroblast (102). It has been shown that mice deficient in JAK1 exhibited severely impaired thymic development and no hematopoietic colony formation in response to IL-7 (103).

- STATs: STATs (Signal Transducer and Activator of Transcription factors), are a family of transcription factors containing SH2 domains that are involved in cytokine mediated signal transduction through the cytoplasmic region of cell surface receptors.

There are 7 different members (STAT1 to STAT4, STAT5A, STAT5B and STAT6) in the STATs family which are activated through JAK.

It has been shown that IL-7 can activate STAT1 and STAT3 (104), but animals deficient in STAT1, 2, or 3 do not show defects in thymocyte development. The SH2 domain of STAT5 docks at tyrosine 449 of the IL-7RD to start the signaling pathway.

STAT5 signaling pathway is known for its anti-apoptotic activity which is exerted through the regulation of expression of several Bcl-2 family members and caspases. It has been shown that IL-7 is required for the survival and development of T lymphocytes, IL-7 is also required for the survival of pre and pro B cells by inducing

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the expression of the anti-apoptotic factors Bcl-2, Bcl-xL, Mcl-1 and by reducing the expression of the death proteins Bax, Bad and Bim (65).

1.3.3.2 PI3 kinase pathway

The phosphatidylinositol 3 (PI3) kinase is one of the downstream pathways of IL-7 that regulates cell survival and proliferation of different cell types. The PI3 kinase pathway has been demonstrated to be important for B and T cell development.

In human T cells, JAK3 associated with the p85 subunit of PI3 kinase following IL-7 stimulation leads to induction of PI3 kinase activation by phosphorylating p85. The activation of PI3 kinase is essential for IL-7 mediated survival and proliferation of human T cell precursors (65).

IL-7 induces activation of AKT, a key downstream target of PI3 kinase, in IL-7 dependent mouse thymocyte cell line (105) and human thymocytes (106). In turn, activated AKT phosphorylates the dead protein Bad, and p27. In addition, AKT also regulates an additional dead protein Bim, through the phosphorylation of forkhead transcription factor FoxO3. By regulating the expression of the dead proteins Bad, p27 and Bim, IL-7 is involved in the regulation of cell survival and cell proliferation through the PI3kinase/AKT pathway (65).

1.3.3.3 Src kinase pathway

The Src family protein kinases (SFKs) are membrane- associated non-receptor protein tyrosine kinases that include nine members Src, Lck, Hck, Fyn, Blk, Lyn, Fgr, Yes and Yrk. It has been shown that IL-7 activates Src kinases. IL-7 stimulation of pre- B cell lines leads to the activation of p59fyn and p53lyn (107). Binding of IL-7 to the IL-7R results in the activation of p56lck and p59fyn of Src kinases, but these are unlikely to be the only pathways responsible for the proliferation of activated T cells in response to IL-7 (108). In peripheral T cells, IL-7 provides signals in addition to the TCR signaling pathways mediated by lck/fyn for the cell survival and proliferation (109). However, the IL-7/IL-7RD activation of the Src kinase pathways leads to different degrees of cell proliferation and homeostasis (65).

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1.3.3.4 IL-7 in metabolism

It has been shown that IL-7 participates in the maintenance of cellular metabolic activity through the cellular uptake of glucose. In a IL-7 dependent thymocyte line, glucose uptake was reduced following withdrawal of the cytokine (110). The expression of glucose transporter type 1 (GLUT1), a protein involved in cellular glucose transport, is regulated by the down-stream substrate of PI3 kinase, AKT. IL-7 promotes GLUT1 expression and increases glucose uptake in leukemic T cells through PI3K and ATK(65).

Figure 2. Outline of IL-7/IL-7R signaling pathways. Activation of different down- stream components of the IL-7R pathway can lead either to cell survival and/or proliferation.

1.3.4 IL-7 and IL-7RD in lymphopenic condition

A study on the serum IL-7 levels in children, before and after 8 weeks from bone marrow transplantation, showed a strong inverse correlation between the circulating IL- 7 levels and the absolute number of lymphocytes (111). This inverse correlation trend between the IL-7 levels and number of CD4+ T cells was also observed in children and adult patients receiving cancer therapy and HIV-1 infected patients (94, 112, 113). In

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cancer patients treated with cytotoxic chemotherapy, the circulating IL-7 levels increased following CD4+ T cell depletion induced by chemotherapy; IL-7 serum levels returned to baseline following recovery of CD4+ T cell counts after completion of therapy.

In HIV-1 infected patients, elevated IL-7 level declines as recovery of the CD4+ T cell counts take place after effective ART. The inversed correlation of serum IL-7 levels with the numbers of CD8+ T cells and B cells is weaker as compared to CD4+ T cells (113). Similar relationships were not observed between the number of lymphocytes and other cytokines including IL-2, IL-4, IL-6, IL-2 and IL-15. This suggested that the relationship of IL-7 and lymphocyte number was unique, and reflected the role of IL-7 in regulation of T cell homeostasis, in stimulation of lymphocyte development and maintenance of peripheral T lymphocytes, especially CD4+ T cells.

As previously described, the binding of IL-7 to the IL-7R receptor leads to intracellular signaling. It has been shown that the IL-7RD is down-regulated in some chronic infections such as Epstein-Barr virus (EBV), cytomegalovirus (CMV), hepatitis C virus (HCV) and HIV-1 infection (114, 115). During lymphopenic condition associated with HIV-1 infection, there is a decrease in the IL-7RD expression on both CD4+ and CD8+

T cells (94, 116, 117). It has been shown that T cells expressing low levels of IL-7RD also express lower levels of the anti-apoptotic Bcl-2 molecule as compared with the IL- 7RD-high T cells in the same donors (94).

The mechanisms leading to high level of circulating of IL-7 and IL-7RD down- regulation during HIV-1 infection are not yet clarified. There are however some possible explanations for these phenomena. An “altruistic” hypothesis has been proposed implying that a T cell that has been exposed to IL-7, and received a sufficient survival signal, would cease consuming IL-7 by down-regulating IL-7R expression thus allowing other cells to receive a survival signal (65). Moreover, since in HIV-1 infection the IL-7RD is down-regulated on T lymphocytes, the efficiency of IL-7 on T cells survival decreases due to lower consumption in spite of the high IL-7 levels in circulation (118). Another possible explanation is that during HIV-1 infection, IL-7 may be accumulated in view of the fact that the number of CD4+ T cells is reduced.

The HIV-1 Tat protein acts by inducing IL-7RD down-regulation, even in presence of

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In HIV-1 infection the majority of IL-7RD low T cells are previously activated antigen- specific T cell clones in late differentiation stages. That suggests the chronic antigenic stimulation to provide a driving force for IL-7RD down regulation (119).

1.4 IMMUNE ACTIVATION IN THE GUT MUCOSA DURING HIV-1 INFECTION

The intestinal mucosa plays an important function as an immunological barrier to pathogens of the outside environment, and also permits a peaceful coexistence with the commensal flora. It has been shown that the GI tract is the largest lymphoid organ in the body, with an estimated surface area 200 times larger than that of the skin (120).

The epithelial layer of the GI tract consists of intestinal epithelial cells (IECs) connected by tight junctions, mucus-secreting goblet cells and antimicrobial-peptide- producing Paneth cells. Interspersed throughout the intestinal epithelium are the GALT, including Peyer’s patches in the small intestine and isolated lymphoid follicles in the colon, which contain immunoglobulin (Ig) A-secreting plasma cells. These different cell populations create and support a mucus layer, containing IgA and antimicrobial peptides, which dramatically reduces the number of bacteria at the barrier between the epithelium and lumen (121).

HIV-1 infection causes damage on the GI tract structure. It has been shown that the HIV-1 Tat protein inhibited the glucose uptake of enterocytes and that the HIV-1 gp120 induced increase of calcium concentration in enterocytes leading to a decreased ability of intestinal epithelial cells to maintain the ionic balance. In chronic infection, HIV-1 causes the damage of intestinal epithelial barriers through apoptosis of enterocytes and decreased luminal defensin. Also in the gut there is a massive depletion of CD4+ T cells and a high number of infected CD4+ T cells which release virions continuously.

Due to the damage of the epithelial barrier there is an increase in microbial translocation and an increased permeability of the intestinal epithelial barrier (122).

In acute infection: Studies conducted during the acute phase of SIV infection in rhesus macaques demonstrated a rapid and almost complete loss of CD4+ T cells from the intestinal lamina propria (39). The depletion of CD4+ T cells occurs at all mucosal surfaces examined regardless of the route of infection, and was found to be due to HIV-1 targeting of CCR5+ memory CD4+ T cells which are the largest proportion of mucosal

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CD4+ T cells. It has been shown that 60% of the mucosal memory CD4+ T cells are infected at the peak of viremia during acute phase of SIV infections, and in the infected animals 80% of the infected cells are depleted within 4 days from the infection (123).

During HIV-1 infection in human, it was shown that a substantial level of CD4+ T cell depletion occurs in the GI tract during HIV-1 infection; this pathogenic feature, which occurs preferentially within the CCR5+T cells, can be found both during the early and the chronic phases of HIV-1 infection (37, 38). Patients treated with ART during the early stages of HIV-1 infection showed a more efficient reconstitution of CD4+ T cells in the GI tract than individuals treated with ART during chronic HIV-1 infection (124).

Figure 3. Immune responses in gut and HIV-1 pathogenesis. Acute HIV-1 infection leads to CD4+ T cell death (black) in the gut, which destructs the mucosal barrier and increases microbial translocation. Bacteria and bacterial components stimulate immune cells to produce pro-inflammatory cytokines, which contribute to chronic HIV- 1 infection and immune activation. In turn, chronic immune activation leads to CD4+

(blue) and CD8+ (green) T cells expansion, thus creating more targets for direct infection of CD4+ T cells (red). Chronic immune activation stimulates HIV-1 replication and also results in lymph node fibrosis, which limits lymph node function to support healthy T cell homeostasis. Due to the fibrosis, CD4+ T cells are retained in lymph nodes and become targets for direct HIV-1 infection and die. DCs, dendritic

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Different mechanisms are proposed to explain the loss of T cells in the GI during HIV- 1 infection. Depletion of T cells can be a direct or indirect effect of the infection:

accordingly loss of CD4+ T cells may be mediated by direct infection (34), or caused by an immune-mediated clearance of infected cells, or bystander apoptosis (35) through CD95-CD95L dependent mechanism (38), or a combination of these mechanisms.

During the acute phase of HIV or SIV infection opposite to the depletion of CD4+ T cells, there is an expansion and/or an influx of CD8+ T cells in the GI tract. However, these CD8+ T cells fail to clear the virus or prevent virus replication and dissemination during primary HIV-1 infection (125).

In chronic phase: Immune activation in HIV-1 chronic infection is an almost pathognomic feature and one of the strongest predictors of disease progression. Some of the manifestations of the chronic immune activation occurring during HIV-1 infection are increased T cell turnover, increased frequencies of T cells with activated phenotype and increased serum levels of pro-inflammatory cytokines and chemokines (Fig. 3).

In the GI tract, during chronic HIV-1 infection, a depletion of the CD4+ T cells continues to occur and this process worsens the damage occurring in the immune system during the acute phase of infection (126). The GI mucosal barrier suffers a serious immunological and structural insult from the very early phases of the infection and this process continues during the chronic phase of HIV-1 infection. Damage to the GI tract during HIV-1 infection may result in microbial translocation. Recently, a study has shown that chronically HIV-1 infected individuals have significantly increased levels of plasma LPS as compare to healthy individuals. Microbial products such as LPS, peptidoglycan and bacterial CpG DNA can directly stimulate the innate immune system through the TLRs or other receptors. The study suggested that microbial translocation may be an important mechanism causing systemic immune activation during chronic HIV-1 infection (28).

In chronic HIV-1 infection, there is a robust and often poly-functional CD8+ T cell response which continuously attempts to fight the HIV-1 infection in the GI tract and to control viral replication at certain level. However the CD8+ T cells cannot clear the chronic HIV-1 infection (127).

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1.5 ANTIRETROVIRAL THERAPY AND IMMUNOTHERAPY

1.5.1 Antiretroviral therapy

ART is considered as the best option for HIV viral suppression and for reduction of morbidity and mortality during HIV infection (128, 129). In spite of the fact that current drugs do not eradicate HIV-1 infection, they help to prolong the relatively healthy life of treated patients as compared to patients who do not receive ART. To date, at least 5 ART drug classes have been recommended to be used for the treatment of HIV infection by the World Health Organization (WHO): Nucleoside reverse transcriptase inhibitors (NRTIs), Nucleotide reverse transcriptase inhibitors (NtRTIs), Non-nucleoside reverse transcriptase inhibitors (NNRTIs), Proteases inhibitors (PIs) and Integrase strand transfer inhibitors (INSTIs) (130). ART is lifelong treatment, and it is currently recommended to use the combination of at least 3 medicines so called highly active antiretroviral therapy (HAART).

Since HIV is genetically highly variable and the patients require lifelong treatment with ART, the major problems with the current therapy are the emergence of drug resistant HIV strains and side effects. In addition, HIV therapy is very expensive and patients under ART treatment need to be monitored regularly and frequently with costly laboratory tests including determination of CD4+ T cells counts and viral load, rendering ART not easily accessible to all HIV infected patients in developing countries (131).

Under ART, the viral replications is suppressed to a low level and the peripheral blood CD4+ T cell number is increased leading to a certain degree of immune reconstitution.

A study in the GI tract showed that ART resulted in the viral load reduction, an increased number of CD4+ T cells and a modest reduction in the number of apoptotic cells in the rectal tissue (132). However, CD4+ T cell recovery in the tissue was poor and occurred at a much slower rate than the increased of CD4+ T cells in peripheral blood (124, 133).

1.5.2 Immunotherapy

In addition to conventional ART, immunotherapy strategies have been investigated to improve immunological recovery during HIV infection. Several approaches have been

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Adoptive therapy: In a adoptive therapy study, infused antigen-specific CTLs showed the capacity to home to sites of virus replication, to retained lytic functions in vivo, and transiently reduce the levels of circulating, productively infected CD4+ T cells (134).

IL-2 therapy: IL-2, which is produced by activated T cells, induces proliferation and cytokine production in T cells, B cells, NK cells and T regulatory cells (T reg). IL-2 has been extensively studied in phases I-II trials, with the administration of IL-2 by subcutaneous injection twice/per day. IL-2 led to increased CD4+ T cells numbers in HIV-1 infected patients (135-138) and also to a significant increase of the survival of CD4+ T memory cells (139). The studies suggest that IL-2 could help in maintaining the functionality of immune cells important for host defense against new antigens as well as for long-term memory to opportunistic infections. To investigate the roles of IL- 2 in clinical benefits during HIV-1 infection, two large phase III trials were conducted.

The ESPRIT (Evaluation of Subcutaneous Proleukin in a Randomized International Trial) study was conducted in patients with CD4+ T cell count> 350 cells/Pl and the SILCAAT (Subcutaneous, Recombinant, Human IL-2 in HIV-Infected Patients with Low CD4 Counts under Active Antiretroviral Therapy) study in patients with CD4+ T cells between 50-299 cells/Pl. Both studies compared the effect of IL-2 plus ART with ART alone. The primary end-point of both studies was opportunistic disease or death.

The CD4+ T cell counts were significantly higher in the IL-2 treated group as compared to patients included in the control group. However, no clinical benefit of IL-2 was found in either studies since the increase in CD4+ T cells did not reduce the risk of opportunistic infections and death (140).

IL-7 therapy: This cytokine plays an important role in T cell homeostasis, and contributes to T cell development and survival. One prospective open-label, phase I »

IIa trial was conducted to investigate the safety and efficacy of IL-7 administered to HIV-1-infected patients treated with HAART. The trial included 13 HIV-1 infected patient under HAART with CD4+ T cell counts between 100 and 400 cells/Pl and plasma HIV-1 RNA levels < 50 copies/ml. Recombinant human (rh) IL-7 was administered (in presence of HAART) with eight subcutaneous injections of two different doses three times a week for a period of 16 days. The rhIL-7 was well tolerated with minor side effects and induced a sustained increase of naive and central memory CD4+ and CD8+ T cells. The study showed that in lymphopenic HIV-1

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infected patients, rhIL-7 therapy induced a substantial quantitative increase in T cells for 48 weeks (141).

In the phase I prospective randomized placebo-controlled study, AIDS clinical trials group (ACTG), a single subcutaneous dose of rhIL-7 was well tolerated with biological activity leading to increased numbers of circulating CD4+ and CD8+ T cells, predominantly of the central memory phenotype. The number of T reg cells (CD25^high CD127^low) number was not affected by rhIL-7 therapy (142).

The studies showed that rhIL-7 therapy led to a sustained increase of naive and central memory CD4+ and CD8+ T cells, and improved T cell function by inducing IFN-Ȗ

and/or IL-2 in response to HIV antigen; these results suggest that patients may benefit from intermittent therapy with IL-7 in combination with ART.

IL-7 therapy was well tolerated in HIV-1 infected patients. However, there are some points which need to be considered. Experiments conducted in vitro showed that IL-7 may up-regulate HIV-1 replication(143), and can enhance HIV-1 proviral reactivation (144). A transient level of elevated HIV mRNA was observed in plasma in 4 of 8 patients receiving 10 Pg/kg rhIL-7 dose in (141) and 6 of 11 rhIL-7 treated patients in (142). In this latter study, at day 56, HIV viral load returned to <50copies/ml in all except one patient. In addition, in one study (142) the T reg population was studied;

since it was concluded that this population, on the contrary of CD4+ T cells, was not expanded the risk remains that development of autoimmunity or other immune dysregulations may accompany IL-7 treatment.

IL-12, IL-10, and IL-15 therapies: IL-12 stimulates T lymphocytes and NK cells to generate a Th1-type immune response. A randomized phase I study was conducted to assess the effect of rhIL-12 therapy in HIV-1 infected patient under HAART. The result showed that IL-12 was well tolerated but had no effect on T lymphocytes subpopulations, antigen specific immune response or viral load (145). The studies on IL-10 and IL-15 therapy also showed no clinical benefit (146, 147).

HIV vaccines: The development of safe prophylactic and therapeutic vaccines with high efficacy is an important goal for the HIV research field. An ideal HIV vaccine should induce cross-neutralizing antibodies against wild-type R5 virus from different

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vaccine development such as using live attenuated, inactivated, virus-like particles, DNA and recombinant vaccines (149). However, despite intensive research, the development of a good candidate vaccine remains elusive. The challenge of developing a HIV vaccine needs new approaches and require new basic research insights (150).

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2 AIMS OF THE THESIS

The overall aim of thesis is focused on the regulation of production and role of IL-7 in HIV-1 infection. The specific aims of the thesis include:

1. To analyze the relationship between serum IL-7 concentrations and T cell numbers in HIV-1 infected patients with variable degree of immune dysfunction.

2. To evaluate the role of CD28- T lymphocytes in inflammatory conditions and immune activation through the modulation of DC and T cell responses during HIV-1 infection.

3. To investigate the role of IL-1E and IFN-J, cytokines produced during inflammatory conditions, in regulation of IL-7 production by stromal and intestinal epithelial cells.

4. To investigate the mechanism through which high levels of IL-7 lead to up- regulation of CD95 receptor on B cells and increased B cell apoptosis during HIV-1 infection.

5. To analyze the relationship between levels of inflammatory cytokines and IL-7 during HIV-1 infection.

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3 MATERIALS AND METHODS

The materials and methods used in the studies enclosed in this thesis are summarized in the following sections.

3.1 PAPER I

Patients: Serum samples and data on T cells numbers were collected for 19 treatment naïve, chronically HIV-1 infected patients in a study period between 13-56 months during 1983-1987 at the Karolinska University Hospital. In addition, 45 HIV-1 infected patients from a previously characterised long-term nonprogressor (LTNP) cohort (151) together with 16 ART-treated, chronically HIV-1 infected individuals from the San Raffaele Institute (Milan) were included in the study. The ethical commissions at the Karolinska Institutet and San Raffaele Institute approved the studies.

Measurement of IL-7 in serum: IL-7 concentration in serum was determined by the Enzyme-linked immunosorbent assay (ELISA) Quantikine high sensitivity immunoassay (R&D Systems, Minneapolis, MN, USA) according to manufacturer’s recommendations.

Statistical analysis: Statistical analyses were performed with the Sigmastat program (SPSS Inc., Chicago, IL, USA). Linear regression analysis or Spearman rank order correlation was used to analyse the association and correlation between the variables.

IL-7 concentrations in different cohorts were compared using t-test.

3.2 PAPER II

Patients and controls: Blood samples were obtained from 12 HIV-1 infected patients, 9 on combination therapy and 3 treatment naïve. The viral load ranged between <50 and 139 000 copy/ml, and the mean CD4+ T cell count was 474 cells/Pl. Blood was also collected from 8 healthy donors. The ethical commission at the Karolinska Institutet approved the studies.

Cellular studies: The CD28- and CD28+ T cell subsets were purified by cell sorter or magnetic separation from peripheral blood. The monocyte-derived DCs were produced by culturing purified monocytes with Granulocyte-macrophage colony-

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stimulating factor (GM-CSF) and IL-4 for five days. The cell markers were measured by flow cytometry.

Cell proliferation was assessed as it follows. Naive T cells isolated from healthy individuals were stained with carboxyfluorescein (CFSE) and then cultured at the density of 10⁶/ml in the presence of DCs (10⁵/ml) pre-treated for 24 hours with CD28+, CD28+CCR7-, and CD28- T cells. Proliferation of the CFSE-labeled T cells was analyzed after four days of activation using flow cytometry.

The production of cytokines in cell culture supernatants, including IL-12, IL-10 and TNF, was measured by ELISA.

3.3 PAPER III.

Cell lines and culture conditions: The human colon adenocarcinoma epithelial cell line DLD-1 and the human bone marrow stromal cell line HS27 were cultured in RPMI-1640 medium and Dulbecco’s modified Eagle’s medium (DMEM; Sigma, St Louis, MO, USA), respectively, with 2 mM L-glutamine, 1% penicillin–streptomycin and 10% heat-inactivated foetal bovine serum (Sigma) in polystyrene flasks with 5%

CO2 at 37 °C. Both cell lines were obtained from the American Type Culture Collection (ATCC).

The cells were seeded in polystyrene 12-well plates (Corning Incorporated, Corning, NY, USA) with medium at a density of 3 × 10⁵ cells/ml for 36 h prior to each experiment. For the experiments, the cells were washed with phosphate-buffered saline (PBS) and fresh medium was added with or without the cytokines: IL-ȕ

(10 ng/ml), IFN-Ȗ ng/ml), TNF-Į ng/ml), IL-2 (10 ng/ml) or the combination of IL-ȕDQG,)1-Ȗwas added for 6 h (for mRNA expression) or for 24 h (for protein determination).

Flow cytometric analysis of cytokine and chemokine receptors on DLD-1 and HS27 cells: Cell surface markers of DLD-1 and HS27 cells were investigated by staining the cells with different antibodies and analysis by Flow cytometry; the data was analyzed by WinMDI 2.9 software (Joseph Trotter, La Jolla, CA, USA).

Relative quantification of human IL-7 mRNA in cell lines: Cellular RNA was isolated

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First-Strand Bead Kit (GE Healthcare Bio-Sciences Corp, NJ, USA) and random primers (Invitrogen, CA, USA).

Relative quantification real-time polymerase chain reaction (PCR) assay was performed by 7900HT ABI PRISM Sequence Detector System (Applied Biosystems) with the human IL-7 assay on-demand kit (catalogue number Hs00174202_m1) and the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) assay (catalogue number:

4333764F) as an endogenous calibrator (Applied Biosystems, Foster City, CA, USA).

The relative expression levels of IL-7 mRNA in the cells, previously stimulated with different cytokines, were compared to the average of IL-7 mRNA expression levels on non-stimulated cells and normalized to the GAPDH mRNA expression levels by the 2[-Delta Delta C(T)] method.

Expression gene profiles using the Affymetrix microarray platform: Total RNA was harvested from the HS27 cell line at 6 h after stimulation with either IL-ȕRU,)1-Ȗ

or the combination of both cytokines; non-stimulated cells were used as controls.

RNA was harvested from three independent experiments for each type of stimulation (IL-ȕ,)1-ȖRUERWKDQGFRQWUROV+6gene expression profiling was performed by using the whole-genome microarray Human Gene 1.0 ST Affymetrix platform (Affymetrix, Inc., Santa Clara, CA, USA) according to standard manufacture’s protocols. Image analysis was performed using Affymetrix Command Console (AGCC) v 1.1, and downstream processing was performed with Affymetrix Expression Console (EC) v 1.1.

Measurement of IL-7 and chemokines: IL-7 concentration in the culture supernatant was determined by Quantikine HS high-sensitivity ELISA kit (R&D Systems); the detection range of this cytokine in the supernatant was 0.156–10.0 pg/ml. The levels of CCL5, CCL20 and CXCL11 in the HS27 and DLD-1 culture supernatants were also tested by Quantikine HS high-sensitivity ELISA kits (R&D Systems). All samples were run in duplicate.

Statistical analysis: Statistical analyses were performed with Sigmastat software (SPSS Inc., Chicago, IL, USA). Pearson product-moment correlation coefficient test was used to measure the correlation between IL-7 and other cytokines. Student’s t- test was used to compare the mean values of IL-7 mRNA expression in cells or IL-7 concentration in culture supernatants between different treatments.

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3.4 PAPER IV

Blood collection and cell culture: Blood samples were collected from healthy blood donors and from 51 HIV-1 infected patients, 49 men and 2 women. 31 patients were on combination therapy and 20 were not treated. The ethical committee at the Karolinska Institutet approved the studies. PBMCs were separated by Ficoll gradient centrifugation (Lymphoprep, Oslo, Norway). For cell cultures monocytes, T and B lymphocytes were separated using respectively the CD14 human microbeads, the Pan T cell Isolation Kit and B cells isolation kit II (Miltenyi Biotech, Bergisch Gladbach, Germany). The purity of the selected cell populations was 90-97% as measured by flow cytometry. Cells were cultured at a density of 1 x10⁶ cells/ml in RPMI-1640 containing L-glutamine, 10%

FCS and antibiotics.

Flow cytometric analysis: Flow cytometric analysis of stained cells was performed by using a FACS LSR II (Becton Dickinson, San Diego, CA) and the data were analysed with FlowJo v. 8.4.4 software (Tree Star Inc., Ashland, OR).

Apoptosis detection: CD95 mediated apoptosis was triggered by human recombinant CD95 ligand (CD95L) (1mg/ml), cross-linked with 20 mg/ml anti-His antibody (both from R&D System, Minneapolis, MN). FITC-conjugated Annexin V reagent (BD Pharmingen) was used to measure apoptosis according to manufacturer’s instructions.

The fractions of cells stained as Vivid negative-Annexin V positive CD3 positive (T lymphocytes), or CD19 positive (B lymphocytes) were considered as apoptotic cells.

Protein Array: Sorted B cells were incubated in IL-7 treated or non treated T cell supernatants for 30 minutes and the phosphorylation patterns was determined using the Human Phospho-Kinase Array Kit (R&D Systems) according to manufacturer’s instruction.

Detection of IFN-ȖP51$OHYHOV: IFN-Ȗ mRNA levels in T cells were detected by real- time PCR with a 7900 CD95T ABI PRISM Sequence Detector System (Applied Biosystem).

Measurement of cytokine concentrations: IFN-Ȗ in culture supernatants was measured by ELISA (BD Pharmingen). Level of IFN-Ȗ, IL-7 and IL-2 in HIV-1 plasma samples were quantified by Luminex technique with Milliplex^® Map kit, High Sensitivity

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Human Cytokine Immunoassay (Millipore Corporation, 290 Concord Road, Billerica, MA 01821, USA).

Statistical analysis: Statistical analysis was performed using the Prism (version 5.0a for Mac OS X, GraphPad Software, La Jolla, CA) using t test and Spearman test.

3.5 CORRELATION OF IL-7 WITH INFLAMMATORY CYTOKINES IN HIV-1 INFECTED PATIENTS RECEIVING ART THERAPY IN VIETNAM

(UNPUBLISHED RESULTS)

Sample collection: Blood samples were collected from 18 HIV-1 infected individuals, classified as AIDS. at the Tayho District Health Center, Hanoi, Vietnam. Among these patients 11 were males and 7 females; the mean age was 32 years. After obtaining written informed consent, blood was collected from all patients at six time points during 1-year period corresponding to start of treatment and 2 weeks, 1, 3, 6 and 12 months after the start of the treatment. Blood samples from 24 healthy HIV negative individuals, age and sex matched, were also collected. The Hanoi Medical University Review board in Bio-medical Research Ethic approved the study.

CD4+ T cell count: CD4+ T cells were measured by BD FACScount (Becton Dickinson, USA) in all fresh blood samples.

HIV-1 viral load: HIV-1 viral load was determined in plasma by the COBAS TaqMan HIV-1 Test (ROCHE Molecular Systems, Inc., Branchburg, NJ, 08876 USA).

Measurement of cytokine concentrations: The concentration of IL-1E, IL-2, IL-7 and IFN-Ȗ were simultaneous quantified in plasma samples by Luminex technique with Milliplex^® Map kit, High Sensitivity Human Cytokine Immunoassay (Millipore Corporation, 290 Concord Road, Billerica, MA 01821, USA).

Statistical analyses: Statistical analysis was performed with Sigmastat software (SPSS Inc., Chicago, IL, USA) using Pearson product-moment correlation coefficient test, ANOVA on ranks and t test.

REGULATION AND ROLE OF IL-7 PRODUCTION IN HIV-1 INFECTION

From the Department of Microbiology, Tumor and Cell Biology Karolinska Institutet, Stockholm, Sweden